Nathalie Ollivier

Sequential native peptide ligation strategies for total chemical protein synthesis

Total chemical synthesis of proteins is usually achieved by assembling unprotected peptide segments using site-specific and chemoselective native peptide ligation methods. Access to large proteins often requires the assembly of at least three segments due to the current limits of solid phase synthesis of individual peptide segments. The aim of this tutorial review is to present the basic concepts and challenges underlying the design of sequential peptide ligation strategies using solution or solid phase chemistry. A special emphasis is given to C-to-N and N-to-C three-segment assembly strategies, which potentially give access to proteins composed of up to 150 amino acid residues.

Bis(2-sulfanylethyl)amino Native Peptide Ligation

The reaction of a peptide featuring a bis(2-sulfanylethyl)amino (SEA) group on its C-terminus with a cysteinyl peptide in water at pH 7 and 37 °C leads to the chemoselective and regioselective formation of a native peptide bond. This method called SEA ligation enriches the native peptide ligation repertoire available to the peptide chemist. Preparation of an innovative solid support which allows the straightforward synthesis of peptide SEA fragments using standard Fmoc/tert-butyl solid phase peptide synthesis procedures is also described.

A One-Pot Three-Segment Ligation Strategy for Protein Chemical Synthesis†

Protein chemical synthesis by native peptide ligation of unprotected peptide segments is an interesting complement and potential alternative to the use of living systems for producing proteins. Actually, tremendous efforts are focused on the design of one-pot strategies allowing the assembly of three peptide segments. The goal is to get rapid access to small proteins (less than 150 amino acid residues), while saving intermediate purification steps and obtaining the products in good yield. Such methods are gaining increasing significance for the study of protein function and appear as a potential option for producing various protein-based therapeutics currently under development. To date, proteins were mainly assembled by sequential native chemical ligation (NCL) or extended methodologies in the C-to-N direction (for recent achievements, see Refs. [8, 9]). NCL involves the chemoselective ligation of a Cterminal peptide thioester, usually an alkylthioester, with an N-terminal cysteine (Cys) peptide. The one-pot sequential Cto-N ligation of three peptide segments designed by Kent et al. is increasingly used for synthesizing proteins. Methods that enable the assembly of peptide segments in the reverse N-to-C direction are rare. 11] Fundamentally, the combination of N-to-C and C-to-N assembly techniques is at the basis of the convergent total synthesis of proteins. The general principle of the one-pot assembly of three peptide segments in the N-to-C direction is illustrated in Scheme 1. Ligation of peptide segments A-X and H-Cys-B-Y yields segment A-Cys-B-Y (Scheme 1, ligation 1). Group Y must ideally be inert during ligation 1 or at least be significantly less reactive than group X to avoid oligomerization or cyclization of segment B. Activation of group Y into Y* subsequently allows the ligation with the third segment H-Cys-C (Scheme 1, ligation 2). For designing a one-pot process working in the N-to-C direction, this activation must be carried out in situ after ligation 1 by using reagents compatible with ligation 2. Furthermore, the Y* group must enable an efficient ligation with the Cys segment C. To date only few one-pot strategies have been described that work in the N-to-C direction and enable the coupling of three peptide segments. 5, 12] Fundamentally, these methods, such as kinetically controlled ligation, rely on the differential reactivity of X and Y groups for peptide-bond formation. In other words, the purity of the target polypeptide is highly dependent on the C-terminal residues of A and B segments and more generally on the accessibility of the reactive ends. Clearly, a strategy in which Y is inert during the first ligation step would bypass these limitations and constitute a critical advance. Herein we show that the combination of NCL and SEA ligation (Scheme 1) permitted design of a solution to this important problem. Reaction of a peptide featuring a C-terminal bis(2-sulfanylethyl)amido group, called SEA hereafter (Scheme 1), with a Cys peptide results in the formation of a native peptide bond in water at pH 7. This reaction probably proceeds via Scheme 1. Total protein synthesis by one-pot assembly of three peptide segments in the N-to-C direction. The first step is a native chemical ligation between thioester segment A and Cys segment B, during which the cyclic disulfide SEA acts as a blocked thioester group (SEA = bis(2-sulfanylethyl)amido). Activation of SEA into SEA by reduction with a phosphine and addition of the third Cys segment C triggers the second ligation step.

Synthesis of peptide alkylthioesters using the intramolecular N,S-acyl shift properties of bis(2-sulfanylethyl)amido peptides.

The design of novel methods giving access to peptide alkylthioesters, the key building blocks for protein synthesis using Native Chemical Ligation, is an important area of research. Bis(2-sulfanylethyl)amido peptides (SEA peptides) 1 equilibrate in aqueous solution with S-2-(2-mercaptoethylamino)ethyl thioester peptides 2 through an N,S-acyl shift mechanism. HPLC was used to study the rate of equilibration for different C-terminal amino acids and the position of equilibrium as a function of pH. We show also that thioester form 2 can participate efficiently in a thiol-thioester exchange reaction with 5% aqueous 3-mercaptopropionic acid. The highest reaction rate was obtained at pH 4. These experimental conditions are significantly less acidic than those reported in the past for related systems. The method was validated with the synthesis of a 24-mer peptide thioester. Consequently, SEA peptides 1 constitute a powerful platform for access to native chemical ligation methodologies.

Synthesis of Thiazolidine Thioester Peptides and Acceleration of Native Chemical Ligation

Thiazolidine thioester peptides were synthesized by reacting bis(2-sulfanylethyl)amido peptides with glyoxylic acid at pH 1. A significant increase in Native Chemical Ligation (NCL) rate was observed with thiazolidine thioesters compared to 3-mercaptopropionic acid-thioester analogues. The method is of particular interest for accelerating valine-cysteine peptide bond formation.

One-pot chemical synthesis of small ubiquitin-like modifier protein–peptide conjugates using bis (2-sulfanylethyl)amido peptide latent thioester surrogates

Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO–peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of peptide segments derivatized by thioester or bis(2-sulfanylethyl)amido (SEA) latent thioester functionalities and the one-pot assembly of the SUMO–peptide conjugate by a sequential native chemical ligation (NCL)/SEA native peptide ligation reaction sequence. This protocol also enables the isolation of a SUMO SEA latent thioester, which can be attached to a target peptide or protein in a subsequent step. It is compatible with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, and it gives access to homogeneous, reversible and functional SUMO conjugates that are not easily produced using living systems. The synthesis of SUMO–peptide conjugates on a milligram scale takes 20 working days.

Access to Large Cyclic Peptides by a One-Pot Two-Peptide Segment Ligation/Cyclization Process

The use of the N-acetoacetyl protecting group for N-terminal cysteine residue enabled creation of an efficient and mild one-pot native chemical ligation/SEA ligation sequence giving access to large cyclic peptides.

A novel PEG-based solid support enables the synthesis of >50 amino-acid peptide thioesters and the total synthesis of a functional SUMO-1 peptide conjugate

A bis(2-sulfanylethyl)amino PEG-based resin enabled the synthesis of large (∼50 Aa) SEA or thioester peptides using Fmoc-SPPS. These peptide segments permitted the first total synthesis of a 97 amino-acid long SUMO-1-SEA peptide thioester surrogate and of a functional and reversible SUMO-1 peptide conjugate.

Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters†

Protein total chemical synthesis enables the atom‐by‐atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C‐terminal peptide thioesters with N‐terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc‐SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2‐sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C‐terminal bis(2‐sulfanylethyl)amido peptides and of peptide thioesters derived from 3‐mercaptopropionic acid. Copyright

Synthesis of oligonucleotide–peptide conjugates using hydrazone chemical ligation

An oligonucleotide was functionalized on the solid-phase by a tartaramide moiety, which could be converted efficiently in solution into a glyoxylyl group following a mild periodic oxidation. The glyoxylyl-oligonucleotide was found to be very stable upon storage and was successfully engaged in hydrazone ligation with an α-hydrazino acetyl peptide.